Characterization of in vitro transcriptional responses of dorsal root ganglia cultured in the presence and absence of blastema cells from regenerating salamander limbs

Abstract

During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema-dorsal root ganglia (DRG) co-culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for five days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1,541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function in injury and neurodevelopmental responses observed for mammalian DRG. We then cultured 5-day DRG explants for an additional five days with or without co-cultured blastema cells. On Day 10, we identified 27 genes whose expression in cultured DRG was significantly affected by the presence or absence of blastema cells. Overall, our study established a DRG-blastema in vitro culture system and identified candidate genes for future investigations of axon regrowth, nerve-blastema signaling, and neural regulation of limb regeneration.

Keywords: Mexican axolotl; axolotl; blastema; dorsal root ganglia; limb regeneration; nerve.

Figure 1

Axolotl dorsal root ganglia (DRG) in vitro. (A) A bright field image of a DRG in vitro 24 h after being explanted. Regenerating neural projections (arrows) are observed at the transected end of the nerve trunk. (B) Image of the same DRG after 4 days of culture in vitro. Neurite outgrowth (arrow) from the cut end of the nerve trunk was robust. (C) Fluorescent image of a sagittal section of a DRG−blastema co‐culture. The blastema was placed on the distal cut end of the DRG with the proximal region of the blastema coming into contact with the regenerating neurites. Nerve fibers were visualized by immunostaining of acetylated α‐tubulin (red), and cell nuclei were stained with 4',6‐diamidino‐2‐phenylindole (DAPI) (blue). Proliferating cells were detected with 5‐ethynyl‐2′‐deoxyuridine (EdU) (green). (D) Diagram of DRG−blastema co‐culture set‐up. The DRG and blastema were adhered to the bottom of the cell culture insert with a drop of growth factor reduced matrigel. (E) Immunofluorescence image of a longitudinal section of a DRG in vitro. The DRG was adhered to the cell culture with matrigel. Phosphorylated neurofilaments were visualized by immunostaining with RT97 (red). Cell nuclei were stained with DAPI (blue). Proliferating cells were detected with EdU (green). Scale bars 0.5 mm.

Figure 2

Venn diagram showing the number of upregulated, differentially expressed genes identified between time and treatment contrasts. Substantially more genes were identified as differentially expressed between day 0 and day 5 cultured dorsal root ganglia (DRG) than between day 10 DRG with blastema (10 B) and day 10 DRG without blastema (10 NB).

Figure 3

Venn diagram showing the number of downregulated, differentially expressed genes identified between time and treatment contrasts. Substantially more genes were identified as differentially expressed between day 0 and day 5 cultured dorsal root ganglia (DRG) than between day 10 DRG with blastema (10 B) and day 10 DRG without blastema (10 NB).